U.S. patent application number 10/121799 was filed with the patent office on 2003-10-16 for peptides for detection of antibody to ehrlichia equi.
Invention is credited to O'Connor, Thomas Patrick JR..
Application Number | 20030194756 10/121799 |
Document ID | / |
Family ID | 28790408 |
Filed Date | 2003-10-16 |
United States Patent
Application |
20030194756 |
Kind Code |
A1 |
O'Connor, Thomas Patrick
JR. |
October 16, 2003 |
Peptides for detection of antibody to Ehrlichia equi
Abstract
The invention provides compositions and methods for the
detection and quantification of Ehrlichia equi antibodies and
antibody fragments.
Inventors: |
O'Connor, Thomas Patrick JR.;
(Westbrook, ME) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Family ID: |
28790408 |
Appl. No.: |
10/121799 |
Filed: |
April 12, 2002 |
Current U.S.
Class: |
435/7.32 ;
435/252.3; 435/320.1; 435/69.3; 530/350; 536/23.7 |
Current CPC
Class: |
G01N 2333/29 20130101;
G01N 2469/20 20130101; C07K 14/29 20130101; G01N 33/6854
20130101 |
Class at
Publication: |
435/7.32 ;
435/69.3; 435/252.3; 435/320.1; 530/350; 536/23.7 |
International
Class: |
G01N 033/554; G01N
033/569; C07H 021/04; C07K 014/195; C12P 021/02; C12N 001/21; C12N
015/74 |
Claims
I claim:
1. A composition of matter consisting essentially of an isolated
polypeptide shown in SEQ ID NO:1 or SEQ ID NO:2.
2. The composition of claim 1, further comprising a carrier.
3. The composition of claim 1, wherein the isolated polypeptide is
conjugated to bovine serum albumin.
4. An isolated polynucleotide encoding the isolated polypeptide of
claim 1.
5. A composition of matter consisting essentially of an isolated
polypeptide fragment of at least about 5 contiguous amino acids of
SEQ ID NO:1 or SEQ ID NO:2.
6. An isolated polynucleotide encoding the isolated polypeptide of
claim 5.
7. A method of detecting antibodies specific for Ehrlichia equi
comprising: (a) contacting a polypeptide shown in SEQ ID NO:1, SEQ
ID NO:2 or both SEQ ID NO:1 and SEQ ID NO:2 with a test sample
suspected of comprising antibodies specific for Ehrlichia equi,
under conditions that allow polypeptide/antibody complexes to form;
(b) detecting polypeptide/antibody complexes; wherein the detection
of polypeptide/antibody complexes is an indication that antibodies
specific for Ehrlichia equi are present in the test sample.
8. The method of claim 7, further comprising contacting the
complexes of step (a) with an indicator reagent comprising a signal
generating compound prior to the performance of step (b).
9. The method of claim 7, wherein the antibodies are fragments of
antibodies.
10. The method of claim 7, wherein the amount of antibody in a test
sample is determined.
11. The method of claim 7, wherein the polypeptide is attached to a
substrate.
12. The method of claim 7, wherein the polypeptide provided is
shown in SEQ ID NO:1.
13. The method of claim 7, wherein the polypeptide provided is
shown in SEQ ID NO:2.
14. The method of claim 7, wherein the polypeptide is provided in a
multimeric form.
15. The method of claim 7, wherein the test sample is a biological
sample obtained from a mammal.
16. The method of claim 15, wherein the mammal is selected from the
group consisting of humans, cats, horses and dogs.
17. The method of claim 7, wherein the method comprises an assay
selected from the group of assays consisting of a reversible flow
chromatographic binding assay, an enzyme linked immunosorbent
assay, a radioimmunoassay, a hemagglutination assay a western blot
assay, a fluorescence polarization immunoassay and an indirect
immunofluorescence assay.
18. An article of manufacture comprising packaging material and,
contained within the packaging material, a polypeptide shown in SEQ
ID NO:1, SEQ ID NO:2, or both polypeptides shown in SEQ ID NO:1 and
SEQ H) NO:2.
19. The article of manufacture of claim 18, wherein the packaging
material comprises a label that indicates that the one or more
polypeptides can be used for the identification of Ehrlichia equi
infection in a mammal.
20. The article of manufacture of claim 18, wherein the
identification of an Ehrlichia equi infection is done using a
method of detecting antibodies specific for Ehrlichia equi
comprising: (a) contacting a polypeptide shown in SEQ ID NO:1, SEQ
ID NO:2, or both SEQ ID NO:1 and SEQ ID NO:2, with a test sample
suspected of comprising antibodies specific for Ehrlichia equi,
under conditions that allow polypeptide/antibody complexes to form;
(b) detecting polypeptide/antibody complexes; wherein the detection
of polypeptide/antibody complexes is an indication that an
Ehrlichia equi infection is present.
21. A method of diagnosing an Ehrlichia equi infection in a mammal
comprising: (a) obtaining a biological sample from a mammal
suspected of having an Ehrlichia equi infection; (b) contacting a
polypeptide shown in SEQ ID NO:1, SEQ ID NO:2, or both SEQ ID NO:1
and SEQ ID NO:2, with the biological sample under conditions that
allow polypeptide/antibody complexes to form; (c) detecting
polypeptide/antibody complexes; wherein the detection of
polypeptide/antibody complexes is an indication that the mammal has
an Ehrlichia equi infection.
22. The method of claim 21, further comprising contacting the
complexes of step (b) with an indicator reagent comprising a signal
generating compound that generates a measurable signal prior to the
performance of step (c).
23. The method of claim 21, wherein the mammal is selected from the
group consisting of humans, cats, horses and dogs.
24. An antibody that specifically binds to at least one epitope of
an Ehrlichia equi polypeptide, wherein said polypeptide is SEQ ID
NO:1 or SEQ ID NO:2.
25. The antibody of claim 24, wherein the antibody is a monoclonal
antibody.
Description
BACKGROUND OF THE INVENTION
[0001] Granulocytic ehrlichiosis occurs in mammals such as humans,
horses, dogs and cats and is caused by infection of granulocytic
cells with the tick-borne agent Ehrlichia equi (E. equi).
Frequently reported symptoms of granulocytic ehrlichiosis in humans
are leukopenia and thrombocytopenia. Common clinical signs in dogs
and horses are fever and anexoria.
[0002] Indirect immunofluorescence assays (IFA) and enzyme-linked
immunosorbent assays (ELISA) are frequently used as aids in the
diagnosis of diseases caused by E. equi by measuring the binding of
antibody from a patient's blood or serum to infected cells, cell
lysates or purified ehrlichial proteins. However, these assays are
severely limited in usefulness because of sensitivity and
specificity issues directly related to the impure nature of the
antigen used in these tests. Highly purified reagents are needed to
construct more accurate assays. This invention discloses specific
synthetic peptide sequences derived from E. equi that can be used
in place of partially purified proteins, infected cells or cell
lysates.
SUMMARY OF THE INVENTION
[0003] It is an object of the invention to provide methods and
compositions for the detection and quantification of Ehrlichia equi
antibodies and antibody fragments. This and other objects of the
invention are provided by one or more of the embodiments described
below.
[0004] One embodiment of the invention provides a composition of
matter consisting essentially of an isolated polypeptide shown in
SEQ ID NO: 1 or SEQ ID NO:2. The composition can comprise a
carrier. The isolated polypeptide of the composition can be
conjugated to bovine serum albumin. The polypeptide of the
composition can consist essentially of a fragment of at least about
5 contiguous amino acids of SEQ ID NO:1 or SEQ ID NO:2. The
invention also comprises an isolated polynucleotide encoding the
isolated polypeptide of the composition.
[0005] Another embodiment of the invention provides a method of
detecting antibodies specific for Ehrlichia equi. The method
comprises contacting a polypeptide shown in SEQ ID NO: 1, SEQ ID
NO:2 or both SEQ ID NO: 1 and SEQ ID NO:2 with a test sample
suspected of comprising antibodies that are specific for Ehrlichia
equi, under conditions that allow polypeptide/antibody complexes to
form. The polypeptide can be attached to a substrate and can be in
a multimeric form. The test sample can a biological sample obtained
from a mammal, such as a human, cat, horse or dog.
Polypeptide/antibody complexes are detected. The detection of
polypeptide/antibody complexes is an indication that antibodies
specific for Ehrlichia equi are present in the test sample. The
polypeptide/antibody complexes can be contacted with an indicator
reagent comprising a signal generating compound prior to the
detection step. The antibodies can be antibody fragments. The
amount of antibody in a test sample can be determined using this
method. The method can comprise an assay selected from the group of
assays consisting of a reversible flow chromatographic binding
assay, an enzyme linked immunosorbent assay, a radioimmunoassay, a
hemagglutination assay a western blot assay, a fluorescence
polarization immunoassay and an indirect immunofluorescence assay.
Still another embodiment of the invention comprises an article of
manufacture comprising packaging material and, contained within the
packaging material, a polypeptide shown in SEQ ID NO:1, SEQ ID
NO:2, or both polypeptides shown in SEQ ID NO:1 and SEQ ID NO:2.
The packaging material can comprise a label that indicates that the
one or more polypeptides can be used for the identification of
Ehrlichia equi infection in a mammal.
[0006] Even another embodiment of the invention provides a method
of diagnosing an Ehrlichia equi infection in a mammal. The method
comprises obtaining a biological sample from a mammal suspected of
having an Ehrlichia equi infection and contacting a polypeptide
shown in SEQ ID NO:1, SEQ ID NO:2, or both SEQ ID NO:1 and SEQ ID
NO:2, with the biological sample under conditions that allow
polypeptide/antibody complexes to form. The polypeptide/antibody
complexes are detected. The detection of polypeptide/antibody
complexes is an indication that the mammal has an Ehrlichia equi
infection. The polypeptide/antibody complexes can be contacted with
an indicator reagent comprising a signal generating compound prior
to the detection step. The mammal can be a human, cat, horse or
dog.
[0007] Another embodiment of the invention provides an antibody
that specifically binds to at least one epitope of an Ehrlichia
equi polypeptide, wherein said polypeptide is SEQ ID NO:1 or SEQ ID
NO:2. The antibody can be a monoclonal antibody.
[0008] The invention therefore provides methods and compositions
that can be used to detect E. equi antibodies and antibody
fragments with improved sensitivity and specificity.
DETAILED DESCRIPTION OF THE INVENTION
[0009] Immunodominant regions of a P30 protein of E. canis have
previously been identified using phage display technology. See U.S.
patent application Ser. No. 09/765,736 filed Jan. 18, 2001. The
identified sequences exhibited strong homology to sequences for
outer membrane proteins of several isolates of Ehrlichia canis.
Synthetic peptides corresponding to sequences from homologous
regions of several outer membrane proteins have been synthesized
and used in diagnostic assays to detect antibodies and antibody
fragments to E. canis.
[0010] E. equi and E. canis are different species of related
organisms that are classified within different serotypes of the
Ehrlichia group. Polypeptide sequences of Ehrlichia equi were
examined to identify immunodominant regions. Immunodominant
sequences derived from an E. equi membrane protein, GE E8 msp-2,
were identified by comparison to E. canis immunodominant
polypeptides (Murphy et al., Infection and Immunity, vol. 66(8),
pp. 3711-3718 (1998)). The following sequences, which correspond to
amino acids numbers 74 to 99 and 62 to 92 of an E. equi membrane
protein, were identified and used as a basis to synthesize two
synthetic peptides:
1 Peptide I. Amino Acid Nos. 74-99 K D G K S V K L E S H K F D W N
T P D P R I G F K D (SEQ ID NO:1) Peptide II. Amino Acid Nos. 65-92
E T K A V Y P Y L K D G K S V K L E S H K F D W N T P D (SEQ ID
NO:2)
[0011] E. equi Polypeptides
[0012] In one embodiment of the invention, a polypeptide or
fragment thereof is substantially pure. Substantially pure means
that a polypeptide of the invention is substantially free from
other biological molecules. A substantially pure polypeptide is at
least 75%, 80%, 90%, 95%, 97%, 99% or 100% pure by dry weight.
Purity can be measured by a method such as column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis.
[0013] Polypeptides of the invention can also comprise fragments of
the polypeptides shown in SEQ ID NO:1, SEQ ID NO:2. For example,
fragments of polypeptides can comprise at least about 5, 6, 8, 10,
12, 15, 18, 20, 22, 24, or 26 contiguous amino acids of the
polypeptides shown in SEQ ID NO:1 or SEQ ID NO:2.
[0014] Polypeptides of the invention can also comprise conservative
variants of the polypeptides shown in SEQ ID NO:1, SEQ ID NO:2. A
conservative variant is a polypeptide that differs from SEQ ID NO:
1 or SEQ ID NO:2, or a fragment thereof, only in conservative
substitutions, such that the antigenic properties of the
polypeptide are substantially the same as the original polypeptide.
Conservative variants can generally be identified by modifying a
polypeptide sequence of the invention and evaluating the antigenic
activity of the modified polypeptide using, for example, an
immunohistochemical assay, an enzyme-linked immunosorbant assay
(ELISA), a radioimmunoassay (RIA), or a western blot assay. A
variant, including a conservative variant and an antigenically
active variant will bind to an anti-E. equi antibody or antibody
fragment with substantially the same binding specificity of a
polypeptide shown in SEQ ID NO:1 or SEQ ID NO:2. "Binding
specificity" or "specifically binds" means that a polypeptide will
substantially recognize and bind to an anti-E. equi polyclonal or
monoclonal antibody or antibody fragment (e.g., a Fv, single chain
Fv, Fab', or F(ab')2 fragment), but does not substantially bind to
other molecules in a test sample. Specific binding can be tested
using, e.g, an immunohistochemical assay, an enzyme-linked
immunosorbant assay (ELISA), a radioimmunoassay (RIA), or a western
blot assay. Polypeptides of the invention can comprise up to about
1, 2, 3, 5, 6, 10, or 15 conservative amino acid substitutions.
[0015] A conservative substitution is one in which an amino acid is
substituted for another amino acid that has similar properties,
such that one skilled in the art of peptide chemistry would expect
the secondary structure and hydropathic nature of the polypeptide
to be substantially unchanged. In general, the following groups of
amino acids represent conservative changes: (1) ala, pro, gly, glu,
asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu,
met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. That
is, amino acids within each of the groups can be substituted for
another amino acid of the same group.
[0016] A polypeptide of the invention or fragments thereof can
differ from the corresponding sequence in SEQ ID NO:1 or SEQ ID
NO:2 and yet retain substantially the same antigenic activity of
the original polypeptide or fragment. This is an antigenically
active variant or an antigenically active fragment. A conservative
variant is one type of a antigenically active variant or fragment.
Naturally occurring antigenically active variants, such as allelic
variants and non-naturally occurring antigenically active variants,
are included in the invention and can be produced by, for example,
mutagenesis techniques or by direct synthesis.
[0017] An antigenically active variant differs by about, for
example, 1, 2, 3, 5, 6, 10, 15 or 20 amino acid residues from a
polypeptide shown in SEQ ID NO: 1 or SEQ ID NO:2 or a fragment
thereof. Where this comparison requires alignment the sequences are
aligned for maximum homology. Deletions, insertions, substitutions,
repeats, inversions or mismatches are considered differences. The
differences are, preferably, differences or changes at a
non-essential residue or a conservative substitution. The site of
variation can occur anywhere in the polypeptide, as long as the
resulting variant polypeptide is antigenicity substantially similar
to a polypeptide shown in SEQ ID NO:1 or SEQ ID NO:2.
[0018] A polypeptide is an antigenically active variant or
antigenically active fragment if it reacts substantially the same
as a polypeptide shown in SEQ ID NO: 1 or SEQ ID NO:2 in an assay
such as an immunohistochemical assay, an ELISA, an RIA, an IFA, or
a western blot assay, e.g. has about 90-110% of the activity of the
original polypeptide. In one embodiment, the assay is a competition
assay wherein the antigenically active variant polypeptide or
fragment is capable of reducing binding of a polypeptide shown in
SEQ ID NO: 1 or SEQ ID NO:2 to a corresponding reactive antigen or
antibody by about 90, 95, 99, or 100%.
[0019] Therefore, the invention provides antigenically active
variant polypeptides that can be at least 85% identical, more
preferably at least 90% identical, and still more preferably at
least 95%, 96%, 97%, 98%, or 99% identical to a polypeptide
sequence shown in SEQ ID NO: 1 or SEQ ID NO:2.
[0020] Identity or identical means amino acid sequence similarity
and has an art recognized meaning. Sequences with identity share
identical or similar amino acids, where similar amino acids are
preferably conserved amino acids. Thus, a candidate sequence
sharing 90% amino acid sequence identity with a reference sequence
(i.e., SEQ ID NO:1) requires that, following alignment of the
candidate sequence with the reference sequence, 90% of the amino
acids in the candidate sequence are identical to the corresponding
amino acids in the reference sequence, and/or constitute
conservative amino acid changes.
[0021] Sequences are aligned for identity calculations using a
mathematical algorithm, such as the algorithm of Karlin and
Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified
as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA
90:5873-5877. Such an algorithm is incorporated into the XBLAST
programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST
protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences with identity
to the polypeptides of the invention. To obtain gapped alignments
for comparison purposes, Gapped BLAST can be utilized as described
in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST) can be used. Internal
gaps and amino acid insertions in the candidate sequence as aligned
are ignored when making the identity calculation.
[0022] Methods of introducing a mutation into amino acids of a
protein is well known to those skilled in the art. See, e.g.,
Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley
and Sons, Inc. (1994); T. Maniatis, E. F. Fritsch and J. Sambrook,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
laboratory, Cold Spring Harbor, N.Y. (1989). Mutations can also be
introduced using commercially available kits such as
"QuikChange.TM. Site-Directed Mutagenesis Kit" (Stratagene). The
generation of a polypeptide antigenically substantially equivalent
to a polypeptide shown in SEQ ID NO:1 or SEQ ID NO:2 by replacing
an amino acid that does not influence the antigenicity of a
polypeptide of the invention can be accomplished by one skilled in
the art.
[0023] Polypeptides of the invention comprise at least one epitope
that is recognized by an anti-E. equi antibody or fragment. An
epitope is an antigenic determinant of a polypeptide. An epitope
can be a linear, sequential epitope or a conformational epitope.
Epitopes within a polypeptide of the invention can be identified by
several methods. See, e.g., U.S. Pat. No. 4,554,101; Jameson &
Wolf, CABIOS 4:181-186 (1988). For example, a polypeptide of the
invention can be isolated and screened. A series of short peptides,
which together span the entire polypeptide sequence, can be
prepared by proteolytic cleavage. By starting with, for example,
20-mer polypeptide fragments, each fragment can be tested for the
presence of epitopes recognized in, for example, an enzyme-linked
immunosorbent assay (ELISA). In an ELISA assay a polypeptide, such
as a 20-mer polypeptide fragment, is attached to a solid support,
such as the wells of a plastic multi-well plate. A population of
antibodies or antibody fragments are labeled, added to the solid
support and allowed to bind to the unlabeled antigen, under
conditions where non-specific adsorbtion is blocked, and any
unbound antibody and other proteins are washed away. Antibody
binding is detected by, for example, a reaction that converts a
colorless indicator reagent into a colored reaction product.
Progressively smaller and overlapping fragments can then be tested
from an identified 20-mer to map the epitope of interest.
[0024] Preferably, a polypeptide of the invention is synthesized
using conventional peptide synthesizers, which are well known in
the art. A polypeptide of the invention can also be produced
recombinantly. A polynucleotide encoding an Ehrlichia equi
polypeptide can be introduced into an expression vector that can be
expressed in a suitable expression system using techniques well
known in the art. A variety of bacterial, yeast, plant, mammalian,
and insect expression systems are available in the art and any such
expression system can be used. Optionally, a polynucleotide
encoding an E. equi polypeptide can be translated in a cell-free
translation system.
[0025] A polypeptide of the invention can be produced as a fusion
protein that contains other amino acid sequences, such as amino
acid linkers or signal sequences, as well as ligands useful in
protein purification, such as glutathione-S-transferase, histidine
tag, and staphylococcal protein A. More than one polypeptide of the
invention can be present in a fusion protein. The polypeptide can
be fused to the N-terminus or C-terminus of a polypeptide of the
invention.
[0026] A polypeptide of the invention can be synthesized such that
it comprises several repeated E. equi polypeptides. This is a
multimeric polypeptide. These repeated polypeptides can comprise
one specific polypeptide, e.g. the polypeptide shown in SEQ ID
NO:1, repeated 2 or more times. Alternatively, the repeated
polypeptides can comprise one or more copies of an E. equi
polypeptide shown in SEQ ID NO:1 along with one or more copies of
an E. equi polypeptide shown in SEQ ID NO:2. A polypeptide of the
invention can be combined or synthesized with one or more non-E.
equi polypeptides, fragments of polypeptides, or full-length
polypeptides.
[0027] A polypeptide of the invention can be combined with a
carrier. A carrier is a vehicle for a polypeptide of the invention.
Carriers include, for example, excipients, diluents, adjuvants, and
stabilizers. Examples of such stabilizers are proteins such as
serum albumins and gelatin; saccharides such as glucose, sucrose,
lactose, maltose, trehalose, sorbitol, maltitol, mannitol and
lactitol; and buffers which are mainly composed of phosphate or
succinate.
[0028] E. equi Polynucleotides
[0029] Polynucleotides of the invention contain less than an entire
microbial genome and can be RNA or single- or double-stranded DNA.
Preferably, the polynucleotides are purified free of other
components, such as proteins and lipids. The polynucleotides of the
invention encode the polypeptides described above. Polynucleotides
of the invention can also comprise other nucleotide sequences, such
as sequences coding for linkers, signal sequences, heterologous
signal sequences, TMR stop transfer sequences, transmembrane
domains, or ligands useful in protein purification such as
glutathione-S-transferase, histidine tag, and staphylococcal
protein A.
[0030] Polynucleotides can be synthesized in the laboratory, for
example, using an automatic synthesizer. An amplification method
such as PCR can be used to amplify polynucleotides from either
genomic DNA or cDNA encoding the polypeptides.
[0031] If desired, polynucleotides can be cloned into an expression
vector comprising, for example, promoters, enhancers, or other
regulatory elements that drive expression of the polynucleotides of
the invention in host cells. An expression vector can be, for
example, a plasmid, such as pBR322, pUC, or ColE1, or an adenovirus
vector, such as an adenovirus Type 2 vector or Type 5 vector.
[0032] A vector comprising a polynucleotide of the invention can be
transformed into, for example, bacterial, yeast, insect, or
mammalian cells so that the polypeptides of the invention can be
expressed in and isolated from cell culture. Any of those
techniques which are available in the art can be used to introduce
polynucleotides into the host cells. These include, but are not
limited to, transfection with naked or encapsulated nucleic acids,
cellular fusion, protoplast fusion, viral infection, and
electroporation.
[0033] Polynucleotides of the invention can be used to produce
polypeptides of the invention and as, for example, as probes or
primers to detect the presence of E. equi polynucleotides in a
sample, such as a biological sample. The ability of such probes to
specifically hybridize to E. equi polynucleotide sequences will
enable them to be of use in detecting the presence of complementary
sequences in a given sample.
[0034] Methods of Detection
[0035] The methods of the invention can be used to detect
antibodies or antibody fragments specific for E. equi in a test
sample, such as a biological sample, an environmental sample, or a
laboratory sample. A biological sample can include, for example,
sera, blood, cells, plasma, or tissue from a mammal such as a
horse, cat, dog or human. The test sample can be untreated,
precipitated, fractionated, separated, diluted, concentrated, or
purified before combining with a polypeptide of the invention.
[0036] The methods comprise contacting a polypeptide of the
invention with a test sample under conditions that allow a
polypeptide/antibody complex to form. That is, a polypeptide of the
invention specifically binds to an antibody specific for E. equi
located in the sample. The formation of a complex between the
polypeptide and anti-E. equi antibodies in the sample is detected.
In one embodiment of the invention, the polypeptide/antibody
complex is detected when an indicator reagent, such as an enzyme,
which is bound to the antibody, catalyzes a detectable reaction.
Optionally, an indicator reagent comprising a signal generating
compound can be applied to the polypeptide/antibody complex under
conditions that allow formation of a polypeptide/antibody/indicator
complex. The polypeptide/antibody/ind- icator complex is detected.
Optionally, the polypeptide or antibody can be labeled with an
indicator reagent prior to the formation of a polypeptide/antibody
complex. The method can optionally comprise a positive or negative
control.
[0037] Assays of the invention include, but are not limited to
those based on competition, direct reaction or sandwich-type
assays. Assays can use solid phases or substrates or can be
performed by immunoprecipitation or any other methods which do not
utilize solid phases. Where a solid phase or substrate is used, a
polypeptide of the invention is directly or indirectly attached to
a solid support or a substrate such as a microtiter well, magnetic
bead, non-magnetic bead, column, matrix, membrane, fibrous mat
composed of synthetic or natural fibers (e.g., glass or
cellulose-based materials or thermoplastic polymers, such as,
polyethylene, polypropylene, or polyester), sintered structure
composed of particulate materials (e.g., glass or various
thermoplastic polymers), or cast membrane film composed of
nitrocellulose, nylon, polysulfone or the like (generally synthetic
in nature). A preferred substrate is sintered, fine particles of
polyethylene, commonly known as porous polyethylene, for example,
10-15 micron porous polyethylene from Chromex Corporation
(Albuquerque, N. Mex.). All of these substrate materials can be
used in suitable shapes, such as films, sheets, or plates, or they
may be coated onto or bonded or laminated to appropriate inert
carriers, such as paper, glass, plastic films, or fabrics. Suitable
methods for immobilizing peptides on solid phases include ionic,
hydrophobic, covalent interactions and the like.
[0038] Polypeptides of the invention can be used to detect anti-E.
equi antibodies or antibody fragments in assays including, but not
limited to enzyme linked immunosorbent assay (ELISA), western blot,
IFA, radioimmunoassay (RIA), hemagglutination (HA), and
fluorescence polarization immunoassay (FPIA). A preferred assay of
the invention is the reversible flow chromatographic binding assay,
for example a SNAPS assay. See U.S. Pat. No. 5,726,010.
[0039] In one type of assay format, one or more polypeptides can be
coated on a solid phase or substrate. A test sample suspected of
containing an anti-E. equi antibody or fragment thereof is
incubated with an indicator reagent comprising a signal generating
compound conjugated to an antibody or antibody fragment specific
for E. equi for a time and under conditions sufficient to form
antigen/antibody complexes of either antibodies of the test sample
to the polypeptides of the solid phase or the indicator reagent
compound conjugated to an antibody specific for E. equi to the
polypeptides of the solid phase. The reduction in binding of the
indicator reagent conjugated to an anti-E. equi antibody to the
solid phase can be quantitatively measured. A measurable reduction
in the signal compared to the signal generated from a confirmed
negative E. equi test sample indicates the presence of anti-E. equi
antibody in the test sample. This type of assay can quantitate the
amount of anti-E. equi antibodies in a test sample.
[0040] In another type of assay format, one or more polypeptides of
the invention are coated onto a support or substrate. A polypeptide
of the invention is conjugated to an indicator reagent and added to
a test sample. This mixture is applied to the support or substrate.
If E. equi antibodies are present in the test sample they will bind
the polypeptide conjugated to an indicator reagent and to the
polypeptide immobilized on the support. The
polypeptide/antibody/indicator complex can then be detected. This
type of assay can quantitate the amount of anti-E. equi antibodies
in a test sample.
[0041] The formation of a polypeptide/antibody complex or a
polypeptide/antibody/indicator complex can be detected by
radiometric, colormetric, fluorometric, size-separation, or
precipitation methods. Optionally, detection of a
polypeptide/antibody complex is by the addition of a secondary
antibody that is coupled to a indicator reagent comprising a signal
generating compound. Indicator reagents comprising signal
generating compounds (labels) associated with a
polypeptide/antibody complex can be detected using the methods
described above and include chromogenic agents, catalysts such as
enzymes, fluorescent compounds such as fluorescein and rhodamine,
chemiluminescent compounds such as dioxetanes, acridiniums,
phenanthridiniums, ruthenium, and luminol, radioactive elements,
direct visual labels, as well as cofactors, inhibitors, magnetic
particles, and the like. Examples of enzymes include alkaline
phosphatase, horseradish peroxidase, beta-galactosidase, and the
like. The selection of a particular label is not critical, but it
will be capable of producing a signal either by itself or in
conjunction with one or more additional substances.
[0042] Formation of the complex is indicative of the presence of
anti-E. equi antibodies in a test sample. Therefore, the methods of
the invention can be used to diagnose E. equi infection in a
patient.
[0043] The methods of the invention can also indicate the amount or
quantity of anti-E. equi antibodies in a test sample. With many
indicator reagents, such as enzymes, the amount of antibody present
is proportional to the signal generated. Depending upon the type of
test sample, it can be diluted with a suitable buffer reagent,
concentrated, or contacted with a solid phase without any
manipulation. For example, it usually is preferred to test serum or
plasma samples which previously have been diluted, or concentrate
specimens such as urine, in order to determine the presence and/or
amount of antibody present.
[0044] The invention further comprises assay kits (e.g., articles
of manufacture) for detecting anti-E. equi antibodies or antibody
fragments in a sample. A kit or article of manufacture comprises
one or more polypeptides of the invention and means for determining
binding of the polypeptide to E. equi antibodies or antibody
fragments in the sample. A kit can comprise a device containing one
or more polypeptides of the invention and instructions for use of
the one or more polypeptides for the identification of an E. equi
infection in a mammal. The kit can also comprise packaging material
comprising a label that indicates that the one or more polypeptides
of the kit can be used for the identification of E. equi infection.
Other components such as buffers, controls, and the like, known to
those of ordinary skill in art, can be included in such test kits.
The polypeptides, assays, and kits of the invention are useful, for
example, in the diagnosis of individual cases of E. equi infection
in a patient, as well as epidemiological studies of E. equi
outbreaks.
[0045] Polypeptides and assays of the invention can be combined
with other polypeptides or assays to detect the presence of E. equi
along with other organisms. For example, polypeptides and assays of
the invention can be combined with reagents that detect heartworm
and/or Borrelia burgdorferi.
[0046] Monoclonal Antibodies
[0047] The polypeptides of the invention can also be used to
develop monoclonal and/or polyclonal antibodies that specifically
bind to an immunological epitope of E. equi present in the
polypeptides of the invention.
[0048] The antibodies or fragments thereof can be employed in assay
systems, such as a reversible flow chromatographic binding assay,
enzyme linked immunosorbent assay, western blot assay, or indirect
immunofluorescence assay, to determine the presence, if any, of E.
equi polypeptides or antibodies in a test sample. In addition,
these antibodies, in particular monoclonal antibodies, can be bound
to matrices similar to CNBr-activated Sepharose and used for the
affinity purification of specific E. equi proteins from, for
example, cell cultures or blood serum, such as to purify
recombinant and native E. equi antigens and proteins. The
monoclonal antibodies of the invention can also be used for the
generation of chimeric antibodies for therapeutic use, or other
similar applications.
[0049] Monoclonal antibodies directed against E. equi epitopes can
be produced by one skilled in the art. The general methodology for
producing such antibodies is well-known and has been described in,
for example, Kohler and Milstein, Nature 256:494 (1975) and
reviewed in J. G. R. Hurrel, ed., Monoclonal Hybridoma Antibodies:
Techniques and Applications, CRC Press Inc., Boca Raton, Fla.
(1982), as well as that taught by L. T. Mimms et al., Virology
176:604-619 (1990). Immortal antibody-producing cell lines can be
created by cell fusion, and also by other techniques such as direct
transformation of B lymphocytes with oncogenic DNA, or transfection
with Epstein-Barr virus.
[0050] The following are provided for exemplification purposes only
and are not intended to limit the scope of the invention described
in broad terms above. All references cited in this disclosure are
incorporated herein by reference.
EXAMPLES
Example 1
Canine
[0051] Three E. equi antibody positive and three E. equi antibody
negative control canine samples (confirmed by Western blot) were
obtained from the Connecticut Agricultural Experiment Station (New
Haven, Conn.). The positive samples were supplied with E. equi
antibody ELISA titers determined by the Connecticut Agricultural
Experiment Station using an E. equi whole cell lysate as an antigen
source.
[0052] The E. equi ELISA titers and results of the microtiter-plate
based immunoassay were obtained using a mixture (50:50) of the
synthetic peptides shown in SEQ ID NO: 1 and SEQ ID NO:2.
Immunoassay synthetic peptides were immobilized on microtiter
wells. A dilution of the test sample was added to the microtiter
well and unbound antibody was removed by washing. Antibody bound to
the immobilized peptide was detected by reaction with an
anti-species, in this case canine, horseradish peroxidase (HRPO)
conjugate, washing and addition of a HRPO substrate. The optical
density of individual microtiter wells was determined using a
microtiter plate reader. The results are shown in Table 1.
Example 2
Equine
[0053] Three E. equi antibody positive and three E. equi antibody
negative control equine samples (confirmed by Western blot) were
obtained from the Connecticut Agricultural Experiment Station. The
positive samples were supplied with E. equi antibody ELISA titers
determined by the Connecticut Agricultural Experiment Station using
an E. equi whole cell lysate as an antigen source. E. equi ELISA
titers and the results of a microtiter-plate based immunoassay were
obtained using a mixture (50:50) of SEQ ID NO: 1 and SEQ ID NO:2.
The peptide-based assay was performed as described above using an
anti-equine:HRPO conjugate. The results are shown in Table 2.
Example 3
Feline
[0054] Three E. equi antibody positive and three E. equi antibody
negative feline samples were obtained from Dr. Steve Levy, a
Connecticut veterinarian. Samples were confirmed by an
immunofluorescence assay (IFA) at North Carolina State University
using an E. equi whole cell lysate as an antigen source.
[0055] The E. equi titers determined by IFA and results of the
microtiter-plate based immunoassay were obtained using a mixture
(50:50) of SEQ ID NO:1 and SEQ ID NO:2. The peptide-based assay was
performed as described above using an anti-feline:HRPO conjugate.
The results are shown in Table 3.
2TABLE 1 Comparison of ELISA results using E. equi whole cell
lysate as antigen source and E. egui synthetic peptides. Sample E.
equi E. equi Synthetic Peptides ID Species ELISA Titer/Result.sup.1
ELISA OD/Result 2249 Canine 2560/Pos 0.068/Pos 2185 Canine
20480/Pos 0.504/Pos 2292 Canine 10240/Pos 0.342/Pos WY05 Canine Neg
0.034/Neg WY023 Canine Neg 0.036/Neg WY013 Canine Neg 0.031/Neg
.sup.1Connecticut Agricultural Experiment Station
[0056]
3TABLE 2 Comparison of ELISA results using E. equi whole cell
lysate as antigen source and E. equi synthetic peptides. Sample E.
equi E. equi Synthetic Peptides ID Species ELISA Titer/Result.sup.1
ELISA OD/Result HO4a Equine 40960/Pos 0.261/Pos H46 Equine 5120/Pos
0.48/Pos H22 Equine 20480/Pos 0.362/Pos Kent 29 Equine Neg
0.055/Neg Kent 26 Equine Neg 0.056/Neg Kent 30 Equine Neg 0.046/Neg
.sup.1Connecticut Agricultural Experiment Station
[0057]
4TABLE 3 Comparison of IFA results using E. equi whole cell lysate
as antigen source and ELISA using E. equi synthetic peptides Sample
Whole Cell Lysate E. equi Synthetic Peptides ID Species IFA
Titer/Result.sup.2 ELISA OD/Result F8 Feline 2048/Pos 0.678/Pos F15
Feline 2048/Pos 0.848/Pos F19 Feline 64/Pos 0.095/Pos F2 Feline Neg
0.036/Neg F3 Feline Neg 0.037/Neg .sup.2North Carolina State
University
[0058]
Sequence CWU 1
1
2 1 26 PRT Ehrlichia equi 1 Lys Asp Gly Lys Ser Val Lys Leu Glu Ser
His Lys Phe Asp Trp Asn 1 5 10 15 Thr Pro Asp Pro Arg Ile Gly Phe
Lys Asp 20 25 2 28 PRT Ehrlichia equi 2 Glu Thr Lys Ala Val Tyr Pro
Tyr Leu Lys Asp Gly Lys Ser Val Lys 1 5 10 15 Leu Glu Ser His Lys
Phe Asp Trp Asn Thr Pro Asp 20 25
* * * * *